Thromboembolic diseases including venous thromboembolism (VTE) and arterial thrombosis are a leading cause of patient morbidity and mortality worldwide. Annually, VTE alone results in approximately 300,000 and 550,000 deaths in the US and Europe, respectively, and an even larger number of non-fatal events (Heit et al., Blood 106, 267a-267a (2005), (Cohen et al. Thromb. Haemost. 98, 756-764 (2007). 4-Hydroxycoumarin (4HC) type oral anticoagulant drugs have been playing significant roles against thromboembolic diseases. Interestingly, anticoagulant function of 4HC derivatives was initially discovered due to its cause of a fetal animal disease manifesting as internal bleeding of the livestock fed with moldy sweet clover forage (called “sweet clover disease”). Indeed, fermentation of plant materials containing melilotoside by molds causes the formation of 4HC and its derivative dicoumarol. The latter demonstrates the blood anticoagulant property by antagonism of vitamin K and acted as a forerunner of the synthetic anticoagulants typified by warfarin (Murray, R. D. H., Méndez, J. & Brown, S. A. Wiley, Chichester (1982). Warfarin is one of the most prescribed oral anticoagulants worldwide with a $300 million global market in 2008 (Melnikova, I. Nat. Rev. Drug Discov. 8, 353-354 (2009)). Besides, acenocoumarol and phenprocoumon are commonly administered in Europe (Beinema et al. Thromb. Haemost. 100, 1052-1057 (2008)). These drugs share the 4HC core structure but differ in 3-substitution on the pyrone ring, and can be chemically synthesized using 4HC as an immediate precursor (Ivanov et al. Arch. Pharm. (Weinheim) 323, 521-522 (1990)), (Rueping et al. Beilstein J. Org. Chem. 6, 6 (2010)).
In past decades, various strategies were developed to chemically synthesize 4HC using petro-derived chemicals, such as phenol, acetosalicylate, methylsalicylate, or 2′-hydroxyacetophenone as starting materials (Gao et al. Synthetic Commun. 40, 732-738 (2010)). Nevertheless, increasing concerns on petroleum depletion and environmental issues have stimulated greater efforts towards the development of biological processes utilizing renewable resources instead of petro-based chemicals. The convergence of genetics, bioinformatics, and metabolic engineering greatly promoted the engineered biosynthesis of a variety of pharmaceutically important compounds in heterologous microbial hosts, e.g. artemisinic acid (Ro., et al. Nature 440, 940-943 (2006)), taxadiene (Ajikumar et al. Science 330, 70-74 (2010)), caffeic acid (Lin et al. Microb. Cell. Fact. 11, 42 (2012), benzylisoquinoline alkaloids (Nakagawa et al. Nat. Commun. 2 (2011)), terpenoids (Martin et al. Nat. Biotechnol. 21, 796-802 (2003)), anthocyanin (Yan et al. Biotechnol. Bioeng. 100, 126-140 (2008)), flavonoids (Santos et al. Metab. Eng. 13, 392-400 (2011)), and resveratrol (Lim et al. Appl. Environ. Microbiol. 77, 3451-3460 (2011)). All these successful cases were built on thorough understanding of the products' native biosynthetic mechanisms, especially genetic and biochemical properties of the involved enzymes. However, lack of knowledge in these aspects hindered the reconstitution of the biosynthesis of pharmaceutically important 4HC. Although it was proposed that 4HC was formed when melilotoside-containing plant materials were fermented by molds and a biosynthetic scheme was described by isotopic labeling analysis (Lequesne, P. W., J. Am. Chem. Soc. 105, 6536-6536 (1983)), involved enzymes have not been identified (Bye et al. Biochem. J. 117, 237-245 (1970)). Several recent studies revealed that the ortho-hydroxylated cinnamoyl-CoA analogs can form coumarins by spontaneous trans/cis isomerization and lactonization (Kai et al. Plant J. 55, 989-999 (2008)), (Vialart et al. Plant J. 70, 460-470 (2012)), (Matsumoto et al. Phytochemistry 74, 49-57 (2012)), suggesting that the pathway might be shunted from trans-2-coumaroyl-CoA to generate coumarin rather than 4HC. Recently, Liu et al identified several biphenyl synthases (BISs) from Sorbus aucuparia that catalyze the formation of 3,5-dihydroxybiphenyl through decarboxylative condensation of three malonyl-CoA molecules with benzoyl-CoA. Surprisingly, when ortho-hydroxybenzoyl-CoA (salicoyl-CoA) was used in place of benzoyl-CoA as a substrate, only one molecule of malonyl-CoA was condensed to form 4HC, suggesting that the ortho-hydroxyl group facilitates the intramolecular cyclization without the condensation of another two malonyl-CoA molecules. Accordingly, a biosynthetic pathway extended from plant salicylate biosynthesis was proposed (Liu et al. Plant Mol. Biol. 72, 17-25 (2010)). However, the same study reported that S. aucuparia cells cannot produce 4HC natively even with the presence of supplemented salicylate (Liu et al. Plant Mol. Biol. 72, 17-25 (2010)), indicating the absence of a CoA ligase that can convert salicylate to salicoyl-CoA. In addition, salicylate biosynthesis in plants has not been fully elucidated (Chen et al. Plant Signal Behav. 4, 493-496 (2009)).